The present invention relates generally to the fields of infertility, endometrial hyperplasia, assisted reproduction, and hormone replacement therapy for women, and methods of assessing and monitoring endometrial development in connection with diagnoses and therapies related to the same.
Over 10% of reproductive age couples suffer from infertility. While many of these couples are successfully diagnosed and treated for their underlying conditions, nearly 20-25% are found to have no proven cause for their difficulties in achieving a successful pregnancy. Many of these couples further pursue costly procedures using assisted reproductive technology (ART) in an attempt to overcome their unidentified problems. The ART procedures used in the United States are IVF (in vitro fertilization), GIFT (gamete intrafallopian transfer), and ZIFT (zygote intrafallopian transfer). Yet, even with ART, only 29.5% of fresh, nondonor cycles result in pregnancies and only 24% result in live births. Rates vary between 19% and 25% depending on the cause of the infertility and older women generally have lower rates of success. Also, the live birth rate decreases to 18.6% when frozen embryos are used.
Clearly, despite improvements in embryo quality and culture conditions, some women are still unable to become pregnant. Their infertility, as well as the infertility experienced by many women, is likely caused in part by implantation difficulties. Thus, predictors of implantation potential are needed, both to better understand the causes of infertility in women and to improve the efficacy and reliability of embryo transfer. Despite over two decades of ART, no tools for endometrial evaluations exist which can adequately predict whether implantation will occur during any given ART cycle.
The Menstrual Cycle
The normal human menstrual cycle proceeds, on average, over the course of 28 days. However, menstrual cycle lengths in individual women vary considerably. For example, Trelar and his associates at the University of Minnesota found that three years after menarche, the range of menstrual intervals for 90% of the recorded cycles was 20.4 to 47.7 days. Their study, which analyzed 275,947 menstrual intervals recorded by more than 2700 women over extended periods, found similar variations during all periods of a woman""s reproductive life. This variability is generally a reflection of the varying length of the follicular phase as opposed to the luteal phase, which lasts a relatively constant and predictable 14 days. While the length of a woman""s menstrual cycle is rarely the idealized 28 days, investigations of the endometrium are based on this average 28 day cycle.
During the natural menstrual cycle, the normal human endometrium undergoes a hormone dependent cyclical series of changes, which involve the progression of proliferation to differentiation. Following menstruation, the endometrium enters the proliferative phase where, under the influence of estrogen, there is marked proliferation of epithelial and stromal cells as well as an increase in the length and tortuosity of the glands. These processes lead to an increase in endometrial thickness, culminating in a characteristic maximal 10-12 mm thickness at midcycle. Then, the luteinizing hormone (LH) surge on cycle day (CD) 13 causes ovulation to occur on CD 14. At this point the endometrium enters the secretory phase where, under the influence of progesterone, the endometrium differentiates, blood vessel and glandular tortuosity, as well as secretory activity, increases to a maximum and the stroma becomes edematous and increasingly vascular. It is during the middle of the secretory phase (CD 20-22) that the endometrium is best prepared for the trophoblast invasion portion of implantation. Estimates for the exact timing of the receptive period range from CD 17-19 to CD 20-24.
Histologic Evaluation of the Naturally Cycling Endometrium
The Noyes, Hertig and Rock Study
Histologically, there are a series of changes during the menstrual cycle that can be seen in the hematoxylin and eosin (H and E) stained endometrial slide. These changes in the histologic appearance of the endometrium were first described by Noyes, Hertig and Rock in 1950 and further elucidated several years later. In their study, xe2x80x9cDating the Endometrial Biopsyxe2x80x9d, Noyes and co-workers focused on the components which they felt changed most xe2x80x9crapidly, constantly, and characteristicallyxe2x80x9d during the menstrual cycle. Those changes included features such as gland mitoses, pseudostratification of nuclei, basal vacuolization of gland cells, secretion into the gland lumen, the presence of stromal edema, stromal mitoses, and leukocyte infiltration. Based on their study of the temporal progression of each variable through a 28 day menstrual cycle and the cyclical changes in the histologic appearance of the endometrium, Hertig and co-workers proposed a method by which the endometrium could be dated within the menstrual cycle where the endometrium was assigned a date consistent with the most advanced component of the biopsy. They based the physiologic day on the date of ovulation, which they determined by the low end point of the first basal body temperature phase.
While Noyes et al. described a progressive series of changes in endometrial histology, their methods are neither necessarily appropriate for precise dating nor are they necessarily reflective of the normal, fertile endometrium. For example, they found that dating with their method was better correlated with the date of ovulation than with the onset of the next menstrual period. From this they concluded that the histologic evaluation was best suited to be a form of hormone assay intended to give a rough idea of quantitative progesterone effect, thus only indirectly indicating the time of ovulation and thus cycle day. This was a useful tool in 1950 to document ovulationxe2x80x94a tool that is secondary today to the documentation of the luteinizing hormone (LH) surge. Also, their findings were described as being applicable to what was referred to as xe2x80x9cnormalxe2x80x9d endometrium. Subsequent clinical investigators as well as clinical practitioners have come to interpret this to mean normal fertile endometrium. However, most of the biopsies (percentages not given) were selected from sterility studies (from the hospital sterility clinic and the sterility practices of two staff members). Noyes and co-workers felt justified in claiming that these biopsies were representative of normally menstruating women because of an internal hospital report which claimed that 84.3% of the biopsies from these sterility patients were xe2x80x9cnormal.xe2x80x9d
Accuracy of the Noyes, Hertig and Rock Criteria for Endometrial Dating
Despite the limitations in using the Noyes et al. criterion to precisely date the normal, fertile endometrium, dating the endometrium using their criteria is used virtually exclusively by pathologists in both their clinical and experimental work. This reliance on what has come to be seen as the xe2x80x9cgold standardxe2x80x9d dating method is based on early evaluations such as the study by Noyes and Hamen in 1953. In this study, there was a reported agreement between two specially trained gynecopathologists of xc2x11 day in 82% of cases (i.e., if one pathologist assigned a biopsy as day 24, then another would interpret it as day 23, 24 or 25 82% of the time). Correlation was best in the middle and late secretory phases. Also, 80% of the histologic dates were found to be within 2 days of the physiologic date. Physiologic dating was determined by the midcycle change in basal body temperature (BBT) and the next menstrual period (NMP), which were found to bound a consistent 14.1 day secretory phase (standard deviation 1.7 days). In the 20% of cases where histologic and physiologic dates did not correlate the histologic dating tended to be earlier than the menstrual dating.
Although Noyes and Haman concluded from their study that histologically dating the endometrial was both useful and accurate, it is questionable whether a 82% interobserver agreement is adequate for a test meant to give precise dating information. Also, an analysis of the reasons for their reported interobserver variability deserves a second look. For example, in spite of the fact that the reporting pathologists were specially trained to histologically evaluate the endometrium, interobserver variability was reported to have occurred when slightly different criteria were used by the two pathologists. With an easy to use system, such variability should not have occurred. Particular difficulty was encountered when subnuclear glandular vacuoles were found in areas where the stroma appeared to be late secretory. (Glands with sub-nuclear vacuoles are consistent with the appearance of early secretory glands). Using the strict criterion of dating based on the most advanced portion of the biopsy, the presence of immature glands should not have affected the determination of an overall date.
The usefulness and accuracy of the original Hertig and Rock criteria to date the endometrial biopsy is further put into question when dating is done by pathologists not specially trained in gynecopathology or the particular criteria of dating the endometrial biopsy. As opposed to these specially trained professionals, the pathologists who read the endometrial biopsy for practicing clinicians, and whose reports are used both to guide clinical treatments and to serve as data for clinical studies, generally have no special interest in endometrial histology. When used by these individuals, the method has been shown to be imprecise with high levels of both inter and intra observer variability in its application.
Scott et al. investigated the effect of interobserver variation on the histologic dating of the endometrium with five pathologists, only one of whom was a gynecopathologist. In particular they examined the correlation between in-phase (xe2x89xa62 days between physiologic date and endometrial date) and out-of-phase ( greater than 2 days between physiologic date and endometrial date) determinations in order to determine whether an alternate reading would lead to a change in management. Of note is the fact that the determination that a biopsy is greater than two days out of phase leads to a diagnosis of luteal phase defect. This diagnosis is confirmed by a second biopsy and leads to a luteal phase defect work-up. Out-of-phase was defined as a discrepancy of two or more days between histologic dating and the cycle date based on the patient""s next menses. In their study they found that the probability of patient management being changed depending on the individual reading the biopsy was 39%. This, they concluded, was inappropriately high for a diagnostic test. Of note is the fact that they excluded several biopsies from the study which were found to have xe2x80x9csevere dyssynchronyxe2x80x9d between glands and stroma.
Later studies investigating the rates of inter as well as intra observer variability found rates similar to those reported by Scott et al. For example, Gibson et al. reported that 22% of readings done by two pathologists were discordant by  greater than 2 days and two readings done by the same pathologist on the same biopsy were discordant by xe2x89xa72 days 17.6% of the time. Also, using the same criteria as Scott et al., Smith and co-workers found that 30% of the time a clinical management decision may be altered depending upon the interpreter of endometrial biopsy.
Optimizing the Histologic Evaluation of the Human Endometrium
Clearly it is problematic to date the endometrium using the original Hertig and Rock histologic criteria alone. Of particular interest is the fact the difficulties were often seen in both the original evaluation by Noyes and co-workers and by subsequent studies using non-gynecopathologists when there existed glands that appeared histologically delayed relative to the stroma (i.e., there was dyssynchrony between the development of the glands and of the stroma). This confusion over dealing with dyssynchronous biopsies indicates that rather than view the endometrial biopsy as a tool for determining a single date in the menstrual cycle, it may be necessary and appropriate to evaluate the stroma and glands separately based on the established progression of changes that occur in each compartment subsequent to the onset of the progesterone effect.
Biologically it makes sense to evaluate the glands and stroma as separate compartments of the endometrium. The two are of different embryologic origin: the glands are epithelial, the stroma is mesenchymal, and the two are separated by a basal lamina. The two have different endocrine functions: for example, only the stromal cells produce prolactin (PRL) and the stroma, more so than the glands, can make estrogens from androgens. The stroma and glands respond dissimilarly to estradiol and progesterone and each has an independent maturation process. In addition, research with endometrial culture has found that stroma and glands can grow individually in cell culture systems. These gland cultures and stromal cultures respond differently to hormonal therapy with estrogens and progesterone.
In addition to the biological basis for evaluating the development of the glands and stroma independently, there is evidence that such an analysis would be practically useful. For example, studies by several groups examined the endometrial biopsies of women with known fertility and elucidated a method of evaluating the endometrial biopsy by analyzing individual histologic features (morphometric analysis). These studies found that only a few of those features were needed to date the endometrium. Further, dating the endometrium using a morphometric analysisxe2x80x94as opposed to the traditional Hertig and Rock dating methodxe2x80x94was, in fact, found to be more accurate. Accuracy in these studies was determined by the physiologic date relative to the LH surge and the date relative to well-defined hormonal events.
Other studies suggest that dyssynchrony may indicate an inadequate, implantation resistant endometrium. For example, Dockery et al., concerned with reports of inaccuracy in chronologically dating the endometrium, carried out a study utilizing morphometric analysis. In their study, endometrial biopsies of women with unexplained infertility were compared to those with known fertility. They found that by using a morphometric analysis women with unexplained infertility had a significant deviation from the normal range in five of the 14 features. All five features were related to glandular and not the stromal components of the endometrium and represented those biopsies whose glands were delayed relative to the stroma (dyssynchrony). This association between individual histologic features and unexplained infertility was significantly greater than the association found between overall retarded endometrial development (histologic dating) and infertility.
A practical approach for evaluating the endometrium which takes into account the independent development of the glands and stroma is presented by Hendrickson and Kempson. In their article, they give specific descriptions of the glands and stroma on each day of the standard 28 day menstrual cycle. They point out that exact dating can only be done on secretory phase endometrial samples which should be dated relative to the day of ovulation. They summarized their evaluations in a practical and easy to use decision tree for endometrial dating. In their decision tree, early secretory biopsies are distinguished based on the histologic appearance of the glands while mid and late secretory biopsies are identified by the stromal appearance. While the authors assert that the endometrium should be dated to a 48 hour period based on the most advanced portion of the biopsy (consistent with the original Hertig and Rock criteria), their detailed description of the glands and stroma allow their individual dating and thus the determination of dyssynchrony when present.
Endometrial Markers
The power of the histologic evaluation of the endometrium is limited by both the human factor and by the reality that H and E staining does not reveal biochemical cell differentiation events. Work has thus gone into trying to determine useful biochemical markers in order to both better understand and better diagnose endometrial abnormalities. While most of these markers have been investigated in the context of hyperplasia and malignancy investigations, some have also been used in the attempt to evaluate implantation resistant endometrium. Markers used in these infertility studies include estrogen and progesterone receptors, the xcex1vxcex23 integrin, HOXA10, and MAG (Mouse Ascites Golgi).
Some of these markers have shown the potential for being clinically useful. For example, the xcex1vxcex23 integrin is first found to be expressed in CD 19 biopsies (the first day in the presumed window of implantation) and both integrins and their ligands are found in the trophectoderm of the preimplantation blastocyst. This indicates that the xcex1vxcex23 integrin may take part in the mechanisms underlying early events in implantation. Also, abnormalities in xcex1vxcex23 integrin expression have been found in increased rates in states associated with decreased fertility such as endometriosis and hydrosalpinges as well as in women with unexplained infertility and in out-of-phase as opposed to in-phase biopsies. However, subsequent studies by other groups have had results which contradict the Lessey group""s claims. For example, Creus, et al. found no difference in xcex1vxcex23 integrin expression between women with and without endometriosis as well as between women who did and did not become spontaneously pregnant.
Progesterone receptors and assays to detect them are disclosed in Giangrande PH et al, The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding. Mol Cell Biol. 2000 May;20(9):3102-15; Mote PA et al., Colocalization of progesterone receptors A and B by dual immunofluorescent histochemistry in human endometrium during the menstrual cycle. J Clin Endocrinol Metab. 1999 August;84(8):2963-71; Wang H. et al., Progesterone receptor subtype B is differentially regulated in human endometrial stroma., Mol Hum Reprod. 1998 April;4(4):407-12.; Kumar NS et al., Selective down-regulation of progesterone receptor isoform B in poorly differentiated human endometrial cancer cells: implications for unopposed estrogen action. Cancer Research 1998 May 1;58(9):1860-5; Leslie K K et al., Differential expression of the A and B isoforms of progesterone receptor in human endometrial cancer cells. Only progesterone receptor B is induced by estrogen and associated with strong transcriptional activation. Annals of the New York Academy of Sciences 1997 Sep. 26; 828:17-26; and Mote PA et al., Colocalization of progesterone receptors A and B by dual immunofluorescent histochemistry in human endometrium during the menstrual cycle. J. Clin. Endocrinol. Metab. 1999 Aug;84(8):2963-71, which are each incorporated herein by reference.
Another potentially useful marker is MAG, a mucin related moiety found to be expressed by gland cells from CD 5-19. The correlation between its disappearance and the beginning of the window of implantationxe2x80x94as well as previous findings that mucin-lectin interactions take place between the conceptus and the endometrium during early implantationxe2x80x94implicate MAG in possibly having a role in mediating early implantation events. In addition, abnormal MAG mucin expression in natural cycle biopsies was found to predict pregnancy failures and abnormal expression in mock cycles was found to predict pregnancy failure in donor oocyte transfer cycles. U.S. Pat. No. 5,599,680, which is incorporated herein by reference discloses the use of MAG as a marker.
Despite their potential clinical usefulness, most of these markers have practical limitations to their use. For example, the anti-xcex1vxcex23 integrin marker antibody used in the immunohistochemical investigations only works with frozen biopsies and not the more commonly retrieved formalin fixed, paraffin embedded sections. MAG, while it works well in these sections, and can thus be used in the examination of archival material, works only in the biopsies of blood group A individuals. HOXA10, as opposed to both xcex1vxcex23 integrin and MAG, can not be evaluated with immunohistochemistry. Its expression is measured through the level of its RNA in frozen sections and thus can only be determined with special techniques that are not routinely performed in clinical pathology labs.
While a morphometric analysis of the endometrial biopsy provides a more objective measurement of endometrial development than the standard holistic process of assigning the biopsy a single CD, it is not necessarily the best practical solution for improving endometrial assessment as it is performed in clinical practice. The adoption of this system would require a shift in the paradigm used by pathologists to evaluate endometrial morphology. This might not be easily or consistently implemented. Further, even if it was possible to universally effect such a transformation, it would not necessarily be most efficient for a non-gynecopathologist to identify and then quantitate the appearance of dyssynchronous glands. A better solution might be to use markers of endometrial development; these would be both easy to interpret and easy to quantitate.
While markers investigated to date (such as the xcex1vxcex23 integrin, HOXA10, and MAG) have shown promise in being clinically useful, each has a practical limitation. For example, the xcex1vxcex23 integrin can only be used in quick frozen samples and not in the more commonly retrieved formalin fixed, paraffin embedded sections. MAG works only in the biopsies of blood group A individuals. HOXA10 expression is measured through the level of its RNA in rapidly frozen tissue samples and thus can only be determined with special techniques that are not routinely performed in clinical pathology labs. Thus, informative markers are needed which can be used in the formalin fixed, paraffin embedded endometrial biopsies of all patients.
Regulators of Cell Cycle Progression as Endometrial Markers
While most of the biochemical markers investigated for evaluating the endometrium are products of the differentiated cell and thus reflect the cycle of changing differentiated function, it is also reasonable to hypothesize that the factors which regulate these changes in differentiation might be useful as endometrial markers. The endometrium typically undergoes highly regulated proliferation and differentiation changes every month. Passage of the endometrium through the menstrual cycle requires coordinated control of the endometrial cells through the cell cycle. Positive regulators of proliferation would thus be found in the estrogen driven first half of the menstrual cycle while inhibitors of proliferation, which would allow differentiation to occur, would be most commonly expressed in the progesterone supported second half of the menstrual cycle. Evaluation of the expression of these factors could therefore be useful in studying the normal progression of the endometrium through the menstrual cycle as well as to possibly identify endometrium that is abnormally progressing.
A family of these cell cycle regulators are the cyclins, their related cyclin dependent kinases (CDKs), and the cyclin dependent kinase inhibitors (CDKI). The cell""s progression through the mitotic cycle is controlled by the activation and inactivation of, as well as the interactions between, these cyclins, CDKs and CDKIs. Overall, the cyclins positively regulate mitosis; the cyclins activate the CDKs, and the CDKs are inhibited by the CDKIs. CDKs also inhibit CDKIs. For example, the cyclin E-cdk2 complex is known to down regulate the CDKI, p27. Net control of mitosis is based on the relative levels of the cyclins, CDKs and CDKIs.
The G1 transition is controlled by a group of these cyclins, CDKs and CDKIs. Cyclin E and p27 in particular are critical regulators of the G1 to S transition. Cyclin E, a regulatory subunit of CDK-2, is thought to be rate limiting for the G1/S transition during the mammalian cell cycle. Its regulatory control is so strong that qualitative and quantitative alterations in cyclin E protein have been implicated as indicators of worse prognosis in various cancers. Conversely, p27 is a mitotic inhibitor which functions as a negative regulator of G1 progression and has been proposed to function as a possible mediator of TGFxcex2-induced G1 arrest. p27 is a candidate tumor suppressor gene.
Cyclin E and p27 have been shown to be clinically useful in the evaluation of malignant tissues. p27 has reduced expression in endometrial neoplasia and has been shown to have prognostic significance in human cancers. There are also indications that determining the subcellular localization of p27 is helpful. For example, p27 appears to interact with its targets in the cell nucleus and mislocalization of p27 in the cytoplasm might inactivate p27 by sequestering it away from relevant cellular targets. This cytoplasmic mislocalization of p27 has been reported in human tumors and cell lines, thus indicating that when p27 is in the cytoplasm it is inactive. Also, Soucek et al. found that p27 mislocalization in the cytoplasm resulted in a failure of p27 to inhibit the cell cycle, even when overexpressed.
Some previous studies have examined the changes in cyclin E and p27 expression during the menstrual cycle. Their focus was primarily the evaluation of hyperplasia and the effects of hormonal treatment. They found that more gland cells express cyclin E during the proliferative and early secretory phases than during the late secretory phase. Conversely, p27 expression was found to be negligible during the proliferative phase and markedly increased in the secretory phase. Some cyclin E positive secretory cells were found to express p27, suggesting an interaction between the two molecules. In addition, hyperplastic cells were found to have very low levels of p27. Levels were greatly increased after progesterone treatment. All findings indicated that expression changes of cyclins in the endometrium are likely controlled by the steroid hormones estrogen and progesterone.
While these studies confirmed that cyclin E and p27 are useful in distinguishing between the proliferative and secretory phases, their results are not directly useful for the evaluation of endometrial abnormalities such as glandular-stromal dyssynchrony. They did not evaluate the detailed progression of cyclin E and p27 expression nor their subcellular localization. Their biopsies did not necessarily reflect normal, fertile endometrium: while the biopsies came from women with a previous history of pregnancy, they were collected from hysterectomy specimens which were removed for either leiomyoma or carcinoma in situ of the uterine cervix. In addition, the authors either did not investigate or simply didn""t distinguish biopsies which had internal glandular-stromal dyssynchrony and thus didn""t report on the cyclin E and p27 staining patterns associated with this abnormality.
The standard infertility evaluation has classically included an endometrial assessment achieved through a histologic examination of the H and E stained biopsy. While many couples have achieved pregnancy through optimization of other aspects of the reproductive processxe2x80x94including anatomic evaluation, hormonal control, male factor analysis, and fertilization and embryo manipulation with ARTxe2x80x94many women still do not become pregnant. In these, and other cases, implantation failure may underlie their infertility. Thus, an understanding of endometrial development is important to both help understand the reasons for the implantation failure and to facilitate the creation of easy-to-use methods for predicting endometrial receptivity.
Although endometrial assessment is important, and has a critical role in determining the cause of a woman""s infertility, the tools available for this evaluation are limited in both their usefulness and accuracy. For example, the histologic examination, which is the most common evaluation method used by clinicians, is generally performed using the criteria of Noyes et al. where the endometrium is assigned a date consistent with the most advanced portion of the biopsy. Standard protocol involves comparing the reported date with the physiologic date (generally determined by the LH surge if known; otherwise by the next menstrual period). A discrepancy of greater than two days on two occasions is considered to be abnormal and prompts the diagnosis of a Luteal Phase Defect. Reports on the precision of this tool, however, show that it is actually insufficient for such a diagnostic test. Interobserver agreement of xc2x11 day is generally only found to be approximately 80%, even when the evaluation is performed by a trained gynecopathologist. This variation means that patient management can be changed simply depending on the individual reading the biopsy. Scott et al. reported this rate to be 39%. Clearly, a more objective diagnostic test for endometrial adequacy is needed.
As the techniques of assisted reproductive technology become more effective, an increasing number of patients are being identified who remain infertile due to implantation failure. However, there are still a limited number of tools to assess endometrial receptivity. Further, there is evidence that the tool most commonly used (the histologic examination of the H and E stained slide using the traditionally cited Hertig and Rock criteria) is insufficient as an absolute diagnostic tool in its level of detail, its degree of accuracy and its reproducibility.
There is a need to address the problems inherent in histologically evaluating the endometrial biopsy. There is a need for methods of endometrial biopsy evaluation that are easy to do, that use broadly applicable tools, and that provide more accurate and more uniform results than the methods of the past. The present invention is directed to addressing these, and other needs.
The present invention relates to methods of diagnosing an abnormality in endometrial glandular development in a woman suspected of being infertile. The methods comprise the step of detecting expression of cyclin E in the nuclei and/or the cytoplasm of endometrial gland cells in an endometrial tissue sample from on or after day 20 of an idealized 28 day menstrual cycle from a woman suspected of being infertile. Expression of cyclin E in the nuclei of greater than 5% of the gland cells indicates endometrial glandular developmental arrest. Expression of cyclin E of greater than 1+ staining intensity in the cytoplasm of greater than 10% of the gland cells indicates endometrial glandular developmental arrest.
The present invention further relates to methods of predicting abnormal endometrial glandular development. The methods comprise the steps of detecting the level of p27 in the nuclei of cells in a sample of endometrial tissue from day 10-18 of a an idealized 28 day menstrual cycle from a woman suspected of being infertile, and comparing the level of expression with an expected level of expression. Detection of elevated levels of p27 in the sample is predictive that the woman will be diagnosed with endometrial glandular developmental arrest.
The present invention further relates to methods of assessing the suitability of the endometrium for embryo implantation in a woman undergoing ovulation induction. The methods comprise the step of detecting expression of cyclin E in the nuclei and/or the cytoplasm of endometrial gland cells in an endometrial tissue sample from before day 17 of an idealized 28 day menstrual cycle. Expression of cyclin E in the nuclei of greater than 5% of the gland cells indicates the endometrium is unsuitable for embryo implantation. Expression of cyclin E of 2-3+ staining intensity in the cytoplasm of less than 50% of the gland cells indicates the endometrium is unsuitable for embryo implantation.
The present invention further relates to methods of evaluating the effect of a hormonal protocol on endometrial glandular development in a woman undergoing a hormonal protocol to produce a mock cycle. The methods comprise the step of detecting expression of cyclin E in the nuclei and/or the cytoplasm of endometrial gland cells in an endometrial tissue sample from on or after day 20 of an idealized 28 day menstrual cycle from a woman undergoing a hormonal protocol to produce a mock cycle. Expression of cyclin E in the nuclei of greater than 5% of the gland cells indicates endometrial glandular developmental arrest. Expression of cyclin E of greater than 1+ staining intensity in the cytoplasm of greater than 10% of the gland cells indicates endometrial glandular developmental arrest.
The present invention further relates to methods of evaluating a hormone replacement therapy protocol in a woman undergoing hormone replacement therapy. The method comprises the steps of detecting expression of cyclin E in the nuclei and/or the cytoplasm of endometrial gland cells in an endometrial tissue sample from said woman; and detecting expression of p27 in the nuclei an of endometrial gland cells in serial section of said endometrial tissue sample. Expression of cyclin E in the nuclei of greater than 5% of the gland cells indicates excessive estrogen. Expression of cyclin E of greater than 1+ staining intensity in the cytoplasm of greater than 10% of the gland cells indicates excessive estrogen. Expression of p27 in the nuclei of less than 20% of the gland cells indicates deficient progesterone.
The present invention further relates to methods of diagnosing endometrial glandular mitotic arrest in a woman suspected of having endometrial hyperplasia. The methods comprise the steps of detecting expression of cyclin E in the nuclei and/or the cytoplasm of endometrial gland cells in an endometrial tissue sample from said woman and detecting expression of p27 in the nuclei an of endometrial gland cells in serial section of said endometrial tissue sample. Expression of cyclin E in the nuclei of less than 10% of the gland cells and expression of p27 in the nuclei of greater than 10% of the gland cells indicates endometrial glandular mitotic arrest.